Calcium reporter gene

Abstract

As a calcium indicator protein having an excellent fluorescent characteristic and calcium reactivity, there is provided DNA in which one of a nucleotide sequence derivative of a calmodulin-binding sequence (ckkap sequence) of calcium/calmodulin-dependent protein kinase kinase and a nucleotide sequence encoding a calcium-binding sequence (CaM sequence) of calmodulin is linked to a 5′ end of a nucleotide sequence encoding a fluorescent protein, and the other nucleotide sequence is linked to a 3′ end of the nucleotide sequence encoding the fluorescent protein. The calcium indicator protein encoded by this DNA, which based on the derivative of the ckkap sequence as a binding domain for the calcium-bound CaM sequence, exhibits a fluorescent characteristic and calcium reactivity superior to those of conventional calcium indicator proteins.

What is claimed is: 
     
       1. A DNA comprising:
 a first nucleotide sequence encoding a calmodulin-binding sequence (ckkap sequence) of calcium/calmodulin-dependent protein kinase kinase, wherein the ckkap sequence is the amino acid sequence set forth in SEQ ID NO: 6; and 
 a second nucleotide sequence encoding a calcium-binding sequence (CaM sequence) of calmodulin, 
 wherein one of the first or second nucleotide sequence is linked to a 5′ end of a nucleotide sequence encoding a fluorescent protein, and the other of the first or second nucleotide sequence is linked to a 3′ end of the nucleotide sequence encoding the fluorescent protein. 
 
     
     
       2. The DNA according to  claim 1 , wherein
 the first nucleotide sequence and the second nucleotide sequence are each linked to the nucleotide sequence encoding the fluorescent protein via a nucleotide sequence encoding an amino acid linker. 
 
     
     
       3. The DNA according to  claim 2 , wherein
 the first nucleotide sequence is linked to the 5′ end of the nucleotide sequence encoding the fluorescent protein, and the second nucleotide sequence is linked to the 3′ end of the nucleotide sequence encoding the fluorescent protein, 
 the first nucleotide sequence and the nucleotide sequence encoding the fluorescent protein are linked via a nucleotide sequence encoding an amino acid linker A, and 
 the nucleotide sequence encoding the fluorescent protein and the second nucleotide sequence are linked via a nucleotide sequence encoding an amino acid linker B, and wherein 
 the amino acid linker A is (-Pro-Val-) and the amino acid linker is B (-Thr-Arg-). 
 
     
     
       4. A vector comprising the DNA according to  claim 1 . 
     
     
       5. A protein comprising:
 a first polypeptide sequence comprising a calmodulin-binding sequence (ckkap sequence) of calcium/calmodulin-dependent protein kinase kinase, wherein the ckkap sequence is the amino acid sequence set forth in SEQ ID NO: 6; and 
 a second polypeptide sequence comprising a calcium-binding sequence (CaM sequence) of calmodulin, 
 wherein one of the first or second polypeptide sequence is linked to an N-terminus of a polypeptide sequence comprising a fluorescent protein, and the other of the first or second polypeptide sequence is linked to a C-terminus of the polypeptide sequence comprising the fluorescent protein. 
 
     
     
       6. The protein according to  claim 5 , wherein
 the first polypeptide sequence and the second polypeptide sequence are each linked to the polypeptide sequence comprising the fluorescent protein via an amino acid linker. 
 
     
     
       7. The protein according to  claim 6 , wherein
 the first polypeptide sequence is linked to the N-terminus of the polypeptide sequence comprising the fluorescent protein, and the second polypeptide sequence is linked to the C-terminus of the polypeptide sequence comprising the fluorescent protein, 
 the first polypeptide sequence and the polypeptide sequence comprising the fluorescent protein are linked via an amino acid linker A, and 
 the polypeptide sequence comprising the fluorescent protein and the second polypeptide sequence are linked via an amino acid linker B, and wherein 
 the amino acid linker A is (-Pro-Val-) and the amino acid linker B is (-Thr-Arg-). 
 
     
     
       8. A calcium indicator reagent for measuring an action potential in a cell and/or imaging a calcium ion in a cell, the reagent comprising:
 (i) a DNA comprising a first nucleotide sequence encoding a calmodulin-binding sequence (ckkap sequence) of calcium/calmodulin-dependent protein kinase kinase, wherein the ckkap sequence is the amino acid sequence set forth in SEQ ID NO: 6, and a second nucleotide sequence encoding a calcium-binding sequence (CaM sequence) of calmodulin, wherein one of the first or second nucleotide sequence is linked to a 5′ end of a nucleotide sequence encoding a fluorescent protein, and the other of the first or second nucleotide sequence is linked to a 3′ end of the nucleotide sequence encoding the fluorescent protein, 
 or (ii) a vector comprising the DNA of (i). 
 
     
     
       9. The reagent according to  claim 8 , wherein the cell is a neuron. 
     
     
       10. A vector comprising the DNA according to  claim 2 . 
     
     
       11. A vector comprising the DNA according to  claim 3 .